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Supplementary and expanded descriptions of methods and figure legends for supplementary data

Supplementary Figure S1. Identification and refinement of cancer-specific super-enhancers in human AML. Supplementary Figure S2. Mapping of super-enhancers to genes. Supplementary Figure S3. AML SE profiles show pronounced variation in enhancers related to myeloid differentiation and enable de novo stratification into six distinct epigenomic subgroups. Supplementary Figure S4. Recapitulation of SE-defined clusters in AML data from the TCGA. Supplementary Figure S5 An SE is present at the RARA locus in a subset of AML samples. Supplementary Figure S6 SY-1425 is a selective and potent RARα agonist. Supplementary Figure S7 Comparison of SY-1425 response to RARA expression and enhancer score and the effect of an RARα antagonist. Supplementary Figure S8 SY-1425 in vivo pharmacokinetics. Supplementary Figure S9 SY-1425 survival and tissue effects on AML PDX models. Supplementary Figure S10 SY-1425 shows greater potency than ATRA in vitro and in vivo. Supplementary Figure S11 Induction of markers of myeloid differentiation in RARA-high AML cell lines upon SY-1425 treatment. Supplementary Figure S12 Comparison of SY-1425 expression and molecular response to APL. Supplementary Figure S13 SY-1425 expression and molecular response.

GSEA of transcriptional response to SY-1425 in RARA-high cell lines in perturbation gene sets & GSEA of transcriptional response to SY-1425 in RARA-high cell lines in hallmark/pathways and perturbation gene sets

RNA-seq derived signatures and per-cluster weights for TCGA clustering

Table of median survival in TCGA data by predicted clusters

GREAT analysis of genes near H3K27ac peaks up-regulated by SY-1425 in RARA-high cell lines in perturbation gene sets

Patient sample features including cytogenetic alterations and mutations

Table of Enhancer, mRNA, and SY-1425 EC50 values in AML cell lines

Supplementary Legends for Figures 1-9, Tables 1-3, Methods from WNT11 Expression Is Induced by Estrogen-Related Receptor α and β-Catenin and Acts in an Autocrine Manner to Increase Cancer Cell Migration

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, β-catenin (β-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by β-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or β-cat. Furthermore, silencing of ERRα and β-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/β-cat–dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/β-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and β-cat that influences the migratory capacity of cancer cells. Cancer Res; 70(22); 9298–308. ©2010 AACR.

Supplementary Figures 1-9, Tables 1-3 from WNT11 Expression Is Induced by Estrogen-Related Receptor α and β-Catenin and Acts in an Autocrine Manner to Increase Cancer Cell Migration

Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional (e.g., MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g., loss of RB pathway checkpoint function) mechanisms. SY-5609 is a potent and selective CDK7 inhibitor currently in development in patients with solid tumors (NCT04247126). To assess the potential for SY-5609 development in heme malignancies, we evaluated SY-5609 activity in preclinical models of mantle cell lymphoma (MCL). MCL is an aggressive B cell lymphoma driven by genetic alterations in RB pathway genes (e.g. CCND1, RB1, CDKN2A) and by hyper-activation of B-cell receptor (BCR) signaling leading to activation of NF-kB-dependent transcriptional programs that drive cell proliferation and survival. Our results support evaluation of SY-5609 in MCL patients and highlight the potential for combining SY-5609 with Bruton's Tyrosine Kinase (BTK) inhibitors, inhibitors of BCR signal transduction approved for treatment of MCL. Methods: SY-5609 in vitro antiproliferative activity was assessed in a panel of 7 MCL cell lines by evaluating growth rate inhibition (GR) curve metrics GR 50 (drug concentration at which growth rate is inhibited by 50%) and GR max (maximum depth of response) after 5-days of treatment. SY-5609 pharmacodynamic (PD) responses and combination activity with the BTK inhibitor acalabrutinib were further assessed in MCL cell line Mino-1. PD activity was assessed by measuring transcriptional changes in POLR2A using a NanoString-based PD assay from the SY-5609 solid tumor trial. Combination activity in vitro was assessed by comparing GR curve metrics between acalabrutinib, SY-5609, and acalabrutinib in combination with SY-5609. Expression of CCND1 and E2F1, a key transcriptional regulator of DNA replication commitment and progression, were assayed via western blot. Combination activity in vivo was assessed in mice bearing Mino-1 xenograft tumors by comparing tumor growth rate inhibition (GRI) and body weight change in mice treated with acalabrutinib (15mg/kg, QD) and/or SY-5609 (0.5mg/kg, BID, days 1-7 and 15-21) over 25 days. Results: SY-5609, as a single agent, potently inhibited proliferation of all 7 MCL cell lines tested with a GR 50 geometric-mean of 7 nM (range: 2 to 20 nM) and a GR max geometric-mean of -0.2 (range: -0.6 to 0.2). In MCL cell line Mino-1, SY-5609 induced a dose-dependent increase in POLR2A expression to levels associated with SY-5609 antiproliferative activity in Mino-1 cells in vitro and to levels comparable with those observed in peripheral blood mononuclear cells (PBMCs) collected from SY-5609 solid tumor trial patients. In combination with acalabrutinib, SY-5609 demonstrated synergistic antiproliferative activity in Mino-1 cells in vitro, a dose-dependent decrease in acalabrutinib GR 50 (up to ~2-fold at the highest SY-5609 concentration tested, 5nM), and a dose-dependent increase in GR max (up to ~10-fold). In addition, the combination of SY-5609 and acalabrutinib caused dose-dependent decreases in CCND1 and E2F1 protein expression in vitro, which were not observed with either single agent alone. Finally, in mice bearing Mino-1 xenografts, the combination of SY-5609 and acalabrutinib significantly (p < 0.01) increased tumor GRI relative to each single agent at the doses and regimens tested and was well tolerated (no difference in body weight changes relative to vehicle treated mice). Conclusions: SY-5609 is a potent and selective CDK7 inhibitor that demonstrates antiproliferative activity in MCL cells in vitro, associated with PD changes comparable to those observed in patients enrolled in the SY-5609 solid tumor trial (ENA, 2020). The combination of SY-5609 and acalabrutinib is synergistic in MCL cells in vitro and inhibits expression of key cell cycle regulatory proteins CCND1 and E2F1 at concentrations that are preclinically subtherapeutic for either single agent alone. The combination of SY-5609 and acalabrutinib is also significantly more effective at inhibiting MCL xenograft growth in vivo than either single agent. These results support evaluation of SY-5609, including in combination with BTK inhibitors, in patients with mantle cell lymphoma. Disclosures Johannessen: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Henry: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Sawant: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. D'Ippolito: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Ke: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Lefkowitz: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Eaton: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Dworakowski: Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Rosario: Syros Pharmaceuticals: Current Employment. Hodgson: Syros Pharmaceuticals, Inc: Current Employment, Current equity holder in publicly-traded company.

We characterized the enhancer landscape of 66 patients with acute myeloid leukemia (AML), identifying 6 novel subgroups and their associated regulatory loci. These subgroups are defined by their superenhancer (SE) maps, orthogonal to somatic mutations, and are associated with distinct leukemic cell states. Examination of transcriptional drivers for these epigenomic subtypes uncovers a subset of patients with a particularly strong SE at the retinoic acid receptor alpha (RARA) gene locus. The presence of a RARA SE and concomitant high levels of RARA mRNA predisposes cell lines and ex vivo models to exquisite sensitivity to a selective agonist of RARα, SY-1425 (tamibarotene). Furthermore, only AML patient-derived xenograft (PDX) models with high RARA mRNA were found to respond to SY-1425. Mechanistically, we show that the response to SY-1425 in RARA-high AML cells is similar to that of acute promyelocytic leukemia treated with retinoids, characterized by the induction of known retinoic acid response genes, increased differentiation, and loss of proliferation. Significance: We use the SE landscape of primary human AML to elucidate transcriptional circuitry and identify novel cancer vulnerabilities. A subset of patients were found to have an SE at RARA, which is predictive for response to SY-1425, a potent and selective RARα agonist, in preclinical models, forming the rationale for its clinical investigation in biomarker-selected patients. Cancer Discov; 7(10); 1136–53. ©2017 AACR. See related commentary by Wang and Aifantis, p. 1065.. This article is highlighted in the In This Issue feature, p. 1047

Introduction: Super-enhancer (SE) mapping in non-APL AML patient (pt) blasts identified RARα as a novel therapeutic target in approximately 30% of pts, who have elevated RARA gene expression. It was observed that the enhancer profile of this novel pt segment, where RARA expression was elevated, overlapped with the profile of mature monocytes (McKeown, 2017). Recently, several reports describe AML with monocytic features associated with resistance to venetoclax (Ven), a BCL-2 inhibitor that has emerged as a standard of care for treatment of pts with newly diagnosed (ND) unfit AML in combination with hypomethylating agents (HMAs) (Zhang, 2018; Kuusanmäki, 2019; Pei, 2020). Approximately one-third of pts do not respond to Ven plus HMAs including azacitidine (Aza) (DiNardo, 2019 and 2020), highlighting a continuing significant unmet need in ND unfit AML. SY-1425, a potent and selective RARα agonist, is in development for non-APL AML in combination with Aza and has demonstrated clinical activity with high rates of complete remission (CR) and deep CRs in RARA-positive (RARA+) ND unfit AML (DeBotton, 2019). Based on the overlap of monocytic features with RARA gene expression, we evaluated clinical samples of pts treated with SY-1425 plus Aza to correlate features of Ven resistance with the RARA biomarker and with clinical response to SY-1425 plus Aza. Methods: RARA gene expression in non-APL AML was evaluated in the TCGA and Beat AML RNA-seq datasets. AUC of cell viability curves were used to evaluate ex vivo sensitivity to compounds, including Ven, in the Beat AML dataset. A monocytic expression signature (MES) was developed using the expression of monocytic and primitive RNA markers in the TCGA dataset to analyze the monocytic phenotype. The MES used a logistic regression model with lasso regularization to distinguish FAB M4/5 (monocytic) from FAB M0/1/2 (primitive) using 10-fold cross-validation with 85% sensitivity and 80% specificity. The MES was then applied to the RNA-seq datasets from Beat AML and AML blasts from ND unfit AML pts in the ongoing SY-1425 plus Aza trial (NCT02807558), in which RARA+ pts were determined by an RT-qPCR based biomarker clinical trial assay (CTA). The MES, RARA expression, and Ven resistance-associated features were compared using Spearman's rho correlation; the association of the MES with the RARA biomarker and with IWG clinical responses in SY-1425 plus Aza treated pts was evaluated. Results: Analysis of RNA-seq in TCGA non-APL AML pts demonstrated higher RARA expression in monocytic AML (FAB M4/M5) than primitive AML (FAB M0/M1/M2) (p We further elucidated the relationships of RARA expression, AML monocytic phenotypes, and Ven resistance. Of 121 inhibitors tested ex vivo in primary Beat AML pt samples, Ven was the inhibitor most associated with treatment resistance in RARA+ vs. RARA- samples. Additionally, MES (rho=0.58), RARA (rho=0.48) and BCL-2 expression (rho=-0.49) had similar magnitude of association with ex vivo Ven resistance, with RARA+ samples showing much lower ex vivo sensitivity to Ven than RARA- samples (p=3×10-8). In 12 AML pt samples (Pei, 2020) treated with Ven ± Aza ex vivo, RARA expression was higher in the monocytic leukemia stem cells resistant to Ven ± Aza (p=0.005). To evaluate whether the RARA+ ND unfit AML pts in the ongoing SY-1425 plus Aza clinical trial were enriched for the monocytic phenotype of Ven resistance, RNA-seq was performed on enrolled pt AML blasts. Among 51 treated pts, 86% (19/22) of RARA+ and 83% (24/29) of RARA- pts yielded RNA-seq results. RARA+ pts were more monocytic than RARA- pts, as demonstrated by higher MES (p=0.002), with higher MCL-1 (p=0.001), and lower BCL-2, CD34, and CD117 expression (p=0.03, 8×10-6, 2×10-4, respectively). In pts with the best IWG response of CR/CRi, RARA+ pts (n=10) had higher MES than RARA- pts (n=8) (p=0.008). Conclusion: In ND unfit AML, RARA+ pts, including those with clinical responses to SY-1425 plus Aza, are enriched for monocytic features associated with resistance to Ven. SY-1425 plus Aza shows potential as a novel targeted regimen for the treatment of RARA+ ND unfit AML and warrants further development in this genomically defined subset of AML pts who may be resistant to upfront SOC therapy with Ven. Disclosures Fiore: Syros Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Kelly:Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Volkert:Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Zhou:Incyte: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Madigan:Syros Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Eaton:Syros Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Hodgson:Syros Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Roth:Syros Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Kang-Fortner:Syros Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company.

Following publication of the original article [1], the authors reported the following errors.

Endocrine-resistance remains a major challenge for treatment of breast cancer. Multiple mechanisms for endocrine resistance have been proposed, including altered expression of ER co-regulators such as Retinoic Acid Receptor Alpha (RARα). Furthermore, crosstalk between estradiol and RA signaling is known and upregulation of RARα has been observed in tamoxifen resistance. We propose a novel treatment paradigm for a newly-defined subset of HR+ patients based on our discovery of a super-enhancer (SE) associated with the RARA locus. SEs are large, highly active chromatin regions that pinpoint cancer vulnerabilities. The RARA SE-identified vulnerability can be targeted using the potent, selective, and metabolically stable RARα agonist SY-1425 (tamibarotene). SY-1425 is approved in Japan to treat Acute Promyelocytic Leukemia, has a well-established efficacy and safety profile, and may enhance response to hormonal therapy (HT) in this newly-defined subset of HR+ patients potentially delaying the need for alternate treatment. Tumor samples from 42 breast cancer patients were analyzed across a range of molecular subtypes. We identified an SE linked to the RARA gene in 54.5% of the hormone positive patient samples. RARA SEs predicted sensitivity to SY-1425 in 12 breast cancer cell lines confirming their functional role, and showed a correlation with RARA gene expression. A panel of 37 breast cancer cell lines was tested for SY-1425 anti-proliferative activity and gene expression levels, and identified RARA as the single best predictor of response. Proliferation of RARA-high cells was inhibited by SY-1425 with low nanomolar EC50s. Transcriptional profiling was performed on 4 HR+ and 3 HER2+/HR- breast cancer cell lines and analyzed by GSEA to examine the molecular response to SY-1425. Signatures for growth including E2F, MYC, DNA replication, and cell cycle were significantly downregulated while retinol metabolism and luminal signaling were upregulated. Estrogen signaling was also significantly altered by SY-1425, supporting known crosstalk between RARα and ER. Consistent with differentiation, CYP26A1 and VE-Cadherin were induced and Actin and Ki67 were diminished at relevant concentrations of SY-1425 and could serve as pharmacodynamic markers of response. To test responses to SY-1425 in vivo, two cell line-derived models and two patient-derived breast cancer models (one RARA-high, and one RARA-low each) were treated with SY-1425. SY-1425 inhibited tumor growth in the RARA-high models, but not the RARA-low models (43% versus 0% TGI). Consistent with the observed changes in transcription, SY-1425 in combination with tamoxifen synergistically inhibited proliferation of RARA-high breast cancer cell lines. Although a few clinical studies have investigated the use of ATRA in HR+ breast cancer without success, our results suggest that patient selection based on the RARA SE may predict which HR+ breast cancer patients could derive benefit by adding an RARα agonist to HT. The potential to prolong or increase the clinical effect of anti-estrogen therapy with SY-1425, which has improved potency, selectivity, and PK stability versus ATRA, would be an attractive strategy to explore. Citation Format: McKeown MR, Fiore C, Lee E, Eaton ML, Orlando D, Guenther MG, Collins C, Chen MW, Fritz CC, di Tomaso E. A novel subgroup of estrogen receptor positive breast cancer may benefit from super-enhancer guided patient selection for retinoic acid receptor α agonist treatment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-18.

Tumor-initiating cells (TICs), also termed cancer stem cells (CSCs) are involved in breast cancer chemoresistance, metastasis and disease progression. To pinpoint tumor cell vulnerabilities and transcriptional drivers of therapeutic relevance, we have characterized the triple negative breast cancer (TNBC) CSC transcriptional landscape using epigenome mapping and nucleosome occupancy determination. We identify a set of transcriptional regulators and signaling mediators that enforce the cancer stem cell state and instruct potential therapeutic strategies. The basal epithelial marker, integrin-β4 (ITGB4), can be used to stratify mesenchymal-like triple-negative breast cancer (TNBC) cells into populations of low and high tumor-initiating ability in vivo. We used ChIP-seq to measure H3K27ac occupancy and map the transcriptional enhancers in SUM159 cells segregated into ITGB4HI (High tumor initiating ability) and ITGB4LOW (Low tumor initiating ability) populations. Gene-enhancer linking and comparative analysis of enhancer usage revealed an epigenomically defined set of genes that are candidate drivers of the CSC cell state, including GSK3β, DNA-binding transcription factors and cellular adhesion proteins. To further define the chromatin architecture and transcriptional regulatory circuitry that underlies CSC state, we deployed ATAC-seq (Assay for Transposase-Accessible Chromatin with high throughput sequencing) within ITGB4HI and ITGB4LOW populations. By pairing nucleosome occupancy and transcription factor kinetics, we created enhancer-linked transcriptional regulatory circuitry of these tumor-initiating cells. Together, the isolation of partially mesenchymal ITGB4HI CSCs, coupled with enhancer mapping and distillation of transcriptional regulatory circuitry from these cells enable the identification of cancer vulnerabilities and therapeutic opportunities for high-risk patients with TNBC. Citation Format: Guenther MG, Lambert AW, Chen MW, Fiore C, Eaton M, Orlando D, Bierie B, Weinberg RA, Fritz CC, Olson ER. Epigenomic analysis of cancer stem cell (CSC)-enriched triple-negative breast cancer (TNBC) populations reveals gene regulatory circuitry and novel tumor cell vulnerabilities [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-04-03.

Epigenomic modifications define gene regulatory features that control transcription and disease cell state. Recent studies of these regulatory features have identified large clusters of enhancers, termed super-enhancers, which define key cell identity and disease genes. Using ChIP-seq and RNA-seq analysis, we have discovered Super-enhancers in breast cancer cell line models and in primary tissue and have characterized their roles in establishing tumor cell state. We find that Super-enhancers recapitulate clinical subgroups in both breast cancer cell line models and in invasive ductal carcinoma. Super-enhancer-associated genes encode known and novel therapeutic targets including kinases, phosphatases, chromatin regulators and transmembrane proteins. Such genes include key drivers such as ERRB2 in HER2+ patient samples, ESR1 in estrogen receptor positive samples, and CCND1 in samples of luminal subtype. We describe the biological and disease relevance of Super-enhancer-associated genes in the context of tumor cell state and drug target discovery. Citation Format: Matthew G Guenther, David A Orlando, Matthew L Eaton, Cindy A Collins, Mei Wei Chen, Sneha Solanki, Jakob Loven, Christian C Fritz, Eric R Olson. Super-enhancer analysis defines breast cancer subtype and identifies tumor dependencies [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-06-04.

Objective We explore the role of DNA methylation in Alzheimer's disease (AD). To elucidate where DNA methylation falls along the causal pathway linking risk factors to disease, we examine causal models to assess its role in the pathology of AD. Methods DNA methylation profiles were generated in 740 brain samples using the Illumina HumanMet450K beadset. We focused our analysis on CpG sites from 11 AD susceptibility gene regions. The primary outcome was a quantitative measure of neuritic amyloid plaque (NP), a key early element of AD pathology. We tested four causal models: (1) independent associations, (2) CpG mediating the association of a variant, (3) reverse causality, and (4) genetic variant by CpG interaction. Results Six genes regions (17 CpGs) showed evidence of CpG associations with NP, independent of genetic variation – BIN1 (5), CLU (5), MS4A6A (3), ABCA7 (2), CD2AP (1), and APOE (1). Together they explained 16.8% of the variability in NP. An interaction effect was seen in the CR1 region for two CpGs, cg10021878 (P = 0.01) and cg05922028 (P = 0.001), in relation to NP. In both cases, subjects with the risk allele rs6656401[superscript AT/AA] display more methylation being associated with more NP burden, whereas subjects with the rs6656401[superscript TT] protective genotype have an inverse association with more methylation being associated with less NP. Interpretation These observations suggest that, within known AD susceptibility loci, methylation is related to pathologic processes of AD and may play a largely independent role by influencing gene expression in AD susceptibility loci., National Institutes of Health (U.S.) (Grant K25AG041906), National Institutes of Health (U.S.) (Grant P30AG10161), National Institutes of Health (U.S.) (Grant R01AG15819), National Institutes of Health (U.S.) (Grant R01AG17917), National Institutes of Health (U.S.) (Grant R01AG36042), National Institutes of Health (U.S.) (Grant R01AG36836), National Institutes of Health (U.S.) (Grant U01AG46152)

FACT (facilitates chromatin transcription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation of spt16 results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037 Spt16-suppressed noncoding transcripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.

DNA replication initiates from thousands of start sites throughout the Drosophila genome and must be coordinated with other ongoing nuclear processes such as transcription to ensure genetic and epigenetic inheritance. Considerable progress has been made toward understanding how chromatin modifications regulate the transcription program; in contrast, we know relatively little about the role of the chromatin landscape in defining how start sites of DNA replication are selected and regulated. Here, we describe the Drosophila replication program in the context of the chromatin and transcription landscape for multiple cell lines using data generated by the modENCODE consortium. We find that while the cell lines exhibit similar replication programs, there are numerous cell line-specific differences that correlate with changes in the chromatin architecture. We identify chromatin features that are associated with replication timing, early origin usage, and ORC binding. Primary sequence, activating chromatin marks, and DNA-binding proteins (including chromatin remodelers) contribute in an additive manner to specify ORC-binding sites. We also generate accurate and predictive models from the chromatin data to describe origin usage and strength between cell lines. Multiple activating chromatin modifications contribute to the function and relative strength of replication origins, suggesting that the chromatin environment does not regulate origins of replication as a simple binary switch, but rather acts as a tunable rheostat to regulate replication initiation events.

Elevated expression of the orphan nuclear receptor estrogen-related receptor α (ERRα) has been associated with a negative outcome in several cancers, although the mechanism(s) by which this receptor influences the pathophysiology of this disease and how its activity is regulated remain unknown. Using a chemical biology approach, it was determined that compounds, previously shown to inhibit canonical Wnt signaling, also inhibited the transcriptional activity of ERRα. The significance of this association was revealed in a series of biochemical and genetic experiments that show that (a) ERRα, β-catenin (β-cat), and lymphoid enhancer-binding factor-1 form macromolecular complexes in cells, (b) ERRα transcriptional activity is enhanced by β-cat expression and vice versa, and (c) there is a high level of overlap among genes previously shown to be regulated by ERRα or β-cat. Furthermore, silencing of ERRα and β-cat expression individually or together dramatically reduced the migratory capacity of breast, prostate, and colon cancer cells in vitro. This increased migration could be attributed to the ERRα/β-cat–dependent induction of WNT11. Specifically, using (a) conditioned medium from cells overexpressing recombinant WNT11 or (b) WNT11 neutralizing antibodies, we were able to show that this protein was the key mediator of the promigratory activities of ERRα/β-cat. Together, these data provide evidence for an autocrine regulatory loop involving transcriptional upregulation of WNT11 by ERRα and β-cat that influences the migratory capacity of cancer cells. Cancer Res; 70(22); 9298–308. ©2010 AACR.

An analysis of mRNA expression in T47D breast cancer cells treated with the synthetic progestin R5020 revealed a subset of progesterone receptor (PR) target genes that are enriched for E2F binding sites. Following up on this observation, we determined that PR-B acts in both direct and indirect manners to positively upregulate E2F1 expression in T47D cells. The direct effects of PR on E2F1 expression were confirmed by chromatin immunoprecipitation (ChIP) analysis, which indicated that the agonist-bound receptor was recruited to several enhancer elements proximal to the E2F1 transcript. However, we also noted that cycloheximide partially inhibits R5020 induction of E2F1 expression, indicating that the ligand-dependent actions of PR on this gene may involve additional indirect regulatory pathways. In support of this hypothesis, we demonstrated that treatment with R5020 significantly increases both hyperphosphorylation of Rb and recruitment of E2F1 to its own promoter, thus activating a positive feedback loop that further amplifies its transcription. Furthermore, we established that PR-mediated induction of Krüppel-like factor 15 (KLF15), which can bind to GC-rich DNA within the E2F1 promoter, is required for maximal induction of E2F1 expression by progestins. Taken together, these results suggest a new paradigm for multimodal regulation of target gene expression by PR.

SY-1425 (tamibarotene) is a potent and selective agonist of the transcription factor (TF) retinoic acid receptor alpha (RARα) that is currently being evaluated in a biomarker-directed Ph2 clinical study in AML and MDS. A subset of AML and MDS patients, referred to as RARA-high, has previously been found to have a large enhancer (super-enhancer) at the RARA locus or upregulation of IRF8, a RARα associated TF. Here, we profile the noncoding genome and transcriptional landscape in AML cells to define the circuitry of RARA-high AML characterized by RARα pathway activation. In addition, AML cell lines were profiled with and without SY-1425 treatment to query the perturbations of key regulatory connections by SY-1425. To better understand the regulatory circuitry of the RARα pathway in AML, we profiled RARA-high and RARA-low AML cell lines with a combination of ATAC-seq, H3K27ac ChIP-seq, and transcriptomic profiling. Using the enhancer landscape of these cell lines compared to a set of healthy human immune cells comprising hematopoietic stem and progenitor cells (HSPCs) as well as multiple myeloid lineages, we performed a non-negative least squares (NNLS) regression to determine the hematopoietic lineage components of these AML cell lines. We found that RARA-high cell lines sensitive to SY-1425 (THP-1, OCI-AML3, MV4-11, and EOL-1) tend to be most similar to monocytes, while RARA-low cell lines insensitive to SY-1425 (KG-1a, Kasumi-1, and OCI-M1) are more HSPC or erythroid-like. We then studied the effect of SY-1425 on the differentiation state of these AML cell lines by additionally profiling them with and without treatment. We find that the enhancer landscape of RARA-high AML cell lines was changed by SY-1425 toward one resembling more fully differentiated myeloid cells of different subtypes, including dendritic cells, granulocytic cells, and macrophages. This was in contrast to the well-characterized granulocytic differentiation of APL caused by retinoids. Functional validation by flow cytometry confirmed surface marker changes consistent with the observed epigenomic alterations. Comparable changes were not found in RARA-low AML cell lines. This effect supports the hypothesis that SY-1425 treatment relieves a RARα-mediated differentiation block of RARA-high AML. To understand the individual regulatory connections being modulated in response to SY-1425, we investigated the binding locations of TFs in the RARA-high cell lines with and without SY-1425 treatment. As expected, enhancers and genes bound by RARα were associated with upregulation of transcription in response to SY-1425. TF binding sites for IRF1 and IRF8 were also identified at induced enhancers. Conversely, known factors important for maintaining an immature/proliferative state were found associated with enhancers most deactivated by SY-1425 (as measured by H3K27ac loss). These included RUNX1, CEBP, and members of the FOS/JUN circuit (which were noted previously as a component of the oncogenic RARα circuit in patient samples). Taken together, these results support that the mechanism of action for SY-1425 is through perturbation of myeloid regulatory relationships in RARA-high AML blasts, leading to a more differentiated phenotype. Based on the understanding of AML- and RARα-driven tumor circuitry, we have generated insight into morphologic, lineage marker, and target-gene based measures to be used in conjunction with clinical studies. The ongoing Ph2 study of SY-1425 in AML and MDS (NCT02807558) will explore measurement of these features in patients. This abstract is also being presented as Poster 06. Citation Format: Christopher M. Fiore, Michael R. McKeown, Emily Lee, Matthew L. Eaton, Darren Smith, Kathryn Austgen, Mei Wei Chen, Matthew Guenther, M. Ryan Corces, Ravindra Majeti, Eric Olson, Christian C. Fritz. SY-1425 (tamibarotene), a potent and selective RARα agonist, induces changes in the transcriptional regulatory circuit of AML cells leading to differentiation [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr PR05.

In patients with acute myeloid leukemia (AML) (≥ 60 years) and myelodysplastic syndrome (MDS), the use of hypomethylating agents (HMAs) may extend survival, but cure rates are very low and new treatment approaches are needed. HMAs, such as azacitidine, work by inhibiting DNMT1, leading to depletion of DNA methylation in the tumor cells. Hypomethylation, in turn, leads to the re-expression of genes associated with differentiation and growth arrest. We have recently explored the potent and selective RARα agonist SY-1425 in a genomically defined subset of AML. SY-1425 binds to RARα and causes a transition from repression to strong activation of target genes, thus reprogramming the tumor cells toward terminal maturation in RARA-high AML models, supporting our recently initiated Phase 2 trial in a biomarker-selected subset of AML and MDS (NCT02807558). Based on potential mechanistic synergy, we evaluated SY-1425 in combination with HMAs and identified a synergistic anti-proliferative effect. In RARA-high AML cell lines, but not RARA-low, the combination of SY-1425 with either azacitidine or decitabine showed synergistic anti-proliferative effects on the cells, with combination indices less than 0.5 over a range of concentrations from 0.01 to 100nM of SY-1425 and 0.1 to 1µM of HMAs. SY-1425 and azacitidine were also co-administered to a disseminated patient-derived xenograft (PDX) mouse model of RARA-high AML. The combination demonstrated superior reduction of tumor burden vs either therapy alone, leading to deeper and more durable responses with less than 1% detectable tumor burden. A follow-up study in the RARA-high PDX model investigated different treatment schedules of SY-1425 and azacitidine over a period of 56 days, supporting a regimen that maximizes anti-tumor activity and tolerability. Mechanistic studies using RNA-seq and ChIP-seq in AML cell line models have revealed that while azacitidine had only moderate suppressive or activating effects over a broad set of genes, the addition of SY-1425 in RARA-high models resulted in strong and specific induction of genes bound by RARα. It is hypothesized that azacitidine acts to prime the tumor cells for reprogramming by SY-1425. The loss of methyl-cytosine residues following azacitidine treatment lowers the barrier to SY-1425 mediated gene induction. It was observed that the two agents work cooperatively to promote terminal differentiation and decrease proliferation of the AML tumor cells, with the potential for increased clinical benefit in a subset of AML defined by a RARA super-enhancer. Based on the largely non-overlapping clinical toxicity profiles of azacitidine and SY-1425, supported by the observed tolerability of the combination in preclinical models, these findings provide a strong rationale for a planned study of this combination in biomarker selected, newly diagnosed AML patients. Citation Format: Michael R. McKeown, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian C. Fritz, Eric Olson. SY-1425 (tamibarotene), a selective RARα agonist, shows synergistic anti-tumor activity with hypomethylating agents in a biomarker selected subset of AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3085. doi:10.1158/1538-7445.AM2017-3085

CD38 is a cell surface protein expressed primarily on white blood cells and considered a marker of differentiation initiation. CD38 is involved in the immune system by engaging cross-talk with T and B cells as well as activation of NK cells. In multiple myeloma (MM), a subset of tumor cells have high CD38 expression (CD38hi), which has led to the development of effective anti-CD38 therapeutic antibodies, such as daratumumab (DARA). Thus, cancer cells that express CD38 can be selectively targeted for elimination by the immune system using these therapeutic antibodies. In multiple myeloma, DARA is most effective in patients whose tumor cells are CD38hi. In contrast, CD38 expression in AML tumors is generally observed to be negative (CD38neg) or dim (CD38dim), and DARA has not shown activity in preclinical AML models. We previously reported that SY-1425, a clinical stage RARα agonist with improved pharmacokinetics, potency, and selectivity over pan-retinoic acid agonists, induces differentiation in non-APL AML cell lines and primary patient samples with a RARA super-enhancer associated biomarker (RARA-high). Since CD38 was found to be among the most differentially expressed genes in response to SY-1425, we hypothesized that SY-1425 mediated CD38 induction to levels comparable to MM may sensitize RARA-high AML cells to anti-CD38 therapy. We demonstrate that SY-1425 treatment of four RARA-high AML cell lines and four RARA-high primary AML patient PBMCs induces the CD38hi phenotype, as measured by flow cytometry, similar to that found in the DARA sensitive MM cells. In contrast, we see no induction in RARA-low cell lines. We then demonstrated the activity of the SY-1425 and DARA combination in an ex vivo NK cell co-culture assay. Two RARA-high AML cell lines treated with SY-1425 and DARA were co-cultured with NK cells and monitored for both antibody dependent cell-mediated cytotoxicity (ADCC) and NK cell activation by interferon gamma production. The combination of SY-1425 and DARA led to a six-fold increase in tumor cell death relative to the single agent controls, and 5-10 fold increases in NK cell activation is observed only in the SY-1425 and DARA combination treatment of RARA-high AML cell lines. Neither single agent, when administered alone, resulted in ADCC. Furthermore, a RARA-low AML cell line does not respond in the ADCC assay following the combination treatment due to the lack of CD38 induction in this SY-1425 insensitive line. In summary, we have identified a novel and rational combination treatment approach for a subset of patients with RARA-high AML. By inducing the expression of CD38, SY-1425 in combination with DARA elicits tumor cell death and NK cell activation. Based on these findings, a phase 2 clinical study with SY-1425 in combination with an anti-CD38 antibody is planned in AML using an RARA biomarker patient selection strategy. Citation Format: Kathryn Austgen, Michael R. McKeown, Darren Smith, Emily Lee, Chris Fiore, Matthew L. Eaton, Christian Fritz, Tracey Lodie, Eric Olson. SY-1425, a selective RARα agonist, induces high levels of CD38 expression in RARA-high AML tumors creating a susceptibility to anti-CD38 therapeutic antibody treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2644. doi:10.1158/1538-7445.AM2017-2644

Prior studies have shown that the RARA gene is associated with a super-enhancer (SE) and has upregulated mRNA expression in a subset of AML patients. Furthermore, this has been found to confer increased sensitivity to SY-1425, a potent and selective RARα agonist. We sought to better characterize the cell state and transcription factor circuitry in these RARA-high AML cells. Clustering of 62 primary AML patient samples based on their genome wide SE maps identified six discrete clusters. RARA-high patients partitioned principally into cluster 2, and to a lesser extent 1, suggesting that RARA upregulation is associated with a specific transcription factor (TF) network and cell state. To start unraveling the TF circuitry in the RARA-high cluster, we investigated which other TFs were SE associated with clusters 1 and 2. In particular, interferon regulatory factor 8 (IRF8) was found to be strongly associated with clusters 1 and 2 by SE and mRNA expression, similar to RARA. Moreover, the expression of both genes is correlated in primary patient samples. IRF8 is involved in interferon signaling and previous studies have shown crosstalk between interferon and retinoic acid signaling. Furthermore, aberrant IRF8 pathway signaling is implicated in AML and CML pathogenesis. The tight clustering of RARA and IRF8 in patient subgroups defined by genome wide enhancer maps suggests RARα and IRF8 may form an integrated transcriptional circuit. Indeed, treatment with SY-1425 was found to strongly induce interferon-like gene expression changes in AML cells with high RARA or IRF8 levels, including the tumor suppressive IFN responsive gene IRF1. While RARA-high AML cell line models have been previously shown to respond to SY-1425, we found that models with high IRF8 expression and low levels of RARA were also found to respond to SY-1425. Such IRF8-high, RARA-low AML cell lines showed activation of similar transcriptional pathways as RARA-high cell lines in response to SY-1425 based on GSEA. IRF8-high AML also had comparable low nM EC50 anti-proliferative effects following SY-1425 treatment. In addition, SY-1425 was found to elicit differentiation in both RARA-high and IRF8-high AML cell lines based on flow cytometry. While RARA and IRF8 expression appear correlated, this data suggests that IRF8 levels may predict for sensitivity to SY-1425 in addition to RARA levels, particularly in cases of AML with high IRF8 expression but low RARA levels. Insights derived from enhancer analysis, transcriptional profiling and differentiation response in preclinical models support the recently initiated Phase 2 trial of SY-1425 (NCT02807558) in which we are evaluating the SE based patient selection strategies and gene circuitry derived pharmacodynamics clinical measurements, including differentiation markers, in patients with AML and MDS. Citation Format: Michael R. McKeown, Matthew L. Eaton, Chris Fiore, Emily Lee, Katie Austgen, Darren Smith, M. Ryan Corces, Ravindra Majeti, Christian C. Fritz. AML patient clustering by super-enhancers reveals an RARA associated transcription factor signaling partner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1511. doi:10.1158/1538-7445.AM2017-1511

Summary The mechanisms contributing to transcription-associated genomic instability are both complex and incompletely understood. Although R-loops are normal transcriptional intermediates, they are also associated with genomic instability. Here, we show that BRCA1 is recruited to R-loops that form normally over a subset of transcription termination regions. There it mediates the recruitment of a specific, physiological binding partner, senataxin (SETX). Disruption of this complex led to R-loop-driven DNA damage at those loci as reflected by adjacent γ-H2AX accumulation and ssDNA breaks within the untranscribed strand of relevant R-loop structures. Genome-wide analysis revealed widespread BRCA1 binding enrichment at R-loop-rich termination regions (TRs) of actively transcribed genes. Strikingly, within some of these genes in BRCA1 null breast tumors, there are specific insertion/deletion mutations located close to R-loop-mediated BRCA1 binding sites within TRs. Thus, BRCA1/SETX complexes support a DNA repair mechanism that addresses R-loop-based DNA damage at transcriptional pause sites., Graphical Abstract, Highlights • Endogenous BRCA1 and senataxin (SETX) interact in a BRCA1-driven process • BRCA1/SETX complexes are recruited to R-loop-associated termination regions (TRs) • BRCA1/SETX complexes suppress transcriptional DNA damage arising at nearby R-loops • BRCA1 breast cancers reveal indel mutations near BRCA1 TR binding regions, Transcriptional R-loops represent a potential threat to genome integrity. Hatchi et al. show that BRCA1, in partnership with SETX, is engaged in a DNA repair mechanism that deals with R-loop-associated genomic instability at transcriptional termination pause sites.

Background Structural rearrangements of the genome resulting in genic imbalance due to copy number change are often deleterious at the organismal level, but are common in immortalized cell lines and tumors, where they may be an advantage to cells. In order to explore the biological consequences of copy number changes in the Drosophila genome, we resequenced the genomes of 19 tissue-culture cell lines and generated RNA-Seq profiles. Results Our work revealed dramatic duplications and deletions in all cell lines. We found three lines of evidence indicating that copy number changes were due to selection during tissue culture. First, we found that copy numbers correlated to maintain stoichiometric balance in protein complexes and biochemical pathways, consistent with the gene balance hypothesis. Second, while most copy number changes were cell line-specific, we identified some copy number changes shared by many of the independent cell lines. These included dramatic recurrence of increased copy number of the PDGF/VEGF receptor, which is also over-expressed in many cancer cells, and of bantam, an anti-apoptosis miRNA. Third, even when copy number changes seemed distinct between lines, there was strong evidence that they supported a common phenotypic outcome. For example, we found that proto-oncogenes were over-represented in one cell line (S2-DRSC), whereas tumor suppressor genes were under-represented in another (Kc167). Conclusion Our study illustrates how genome structure changes may contribute to selection of cell lines in vitro. This has implications for other cell-level natural selection progressions, including tumorigenesis. Electronic supplementary material The online version of this article (doi:10.1186/gb-2014-15-8-r70) contains supplementary material, which is available to authorized users.

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease., National Human Genome Research Institute (U.S.) (RC1HG005334), National Human Genome Research Institute (U.S.) (R01HG004037), National Human Genome Research Institute (U.S.) (R01HG004037-S1), National Human Genome Research Institute (U.S.) (RO1NS078839), National Science Foundation (U.S.) (CAREER Award 1254200)

SY-1425, a potent and selective agonist of the retinoic acid receptor RARα, is being investigated in a Ph2 trial in a novel genomically-defined subset of non-APL AML and MDS patients (clinicaltrials.gov NCT02807558). RARa is a nuclear hormone receptor and transcription factor that regulates genes involved in cell differentiation and proliferation. We identified a super-enhancer (SE) at the RARA locus, the gene encoding RARa, in a subset of primary non-APL AML blasts. Preclinical models demonstrated a correlation between the presence of a RARA SE and sensitivity to SY-1425, providing the rationale for clinical investigation. Further research has investigated pharmacodynamics (PD) markers and combinations of drugs to support clinical development of SY-1425. In this study we identified DHRS3mRNA induction as a measure of RARα target engagement with SY-1425. We also demonstrated synergy in preclinical models with SY-1425 and hypomethylating agents. Since RARα is a transcription factor that regulates target genes when bound by a retinoid, we characterized the dynamic expression changes of a panel of RARA enhancer- high and - low non-APL AML cell lines (hereafter referred to as RARA-high and -low) in response to SY-1425 treatment. DHRS3 showed the largest expression increase following treatment in 3 RARA-high cell lines, with a range of 29 to 115 fold. In contrast, there was a much lower DHRS3 induction in 3 RARA-low cell lines (range of 1.6 to 6.1 fold). Induction was found to be both time- and dose-dependent with maximal induction at approximately 6 hours and half maximal induction near the EC50 for the anti-proliferative effect in RARA-high cell lines. DHRS3 encodes dehydrogenase/reductase (SDR family) member 3, a metabolic enzyme involved in maintaining cellular retinol homeostasis and had previously been shown to be induced by retinoids. Thus, DHRS3induction in tumor cells represents a potentially useful PD marker for clinical studies of SY-1425. To better understand the mechanism of induction of DHRS3 by SY-1425 we examined the chromosomal localization of RARα as well as the epigenomic state of the DHRS3 locus by ChIP-seq for RARα and H3K27 acetylation, the latter being an indicator of active enhancers and promoters. In the untreated state, OCI-AML3 (a typical RARA-high AML cell line) was found to have multiple RARα binding sites both within and distal to the DHRS3 gene but minimal H3K27 acetylation. Following treatment with SY-1425, the level of H3K27 acetylation at DHRS3 increased, resulting in the formation of a SE. Moreover, the SE encompassed the RARα binding sites, consistent with the model in which SY-1425 converts RARα into an activator of DHRS3expression. Similar results were seen for the CD38 locus in which SY-1425 treatment increased expression, H3K27 acetylation, and RARα binding. CD38 is a cell surface antigen and marker of myeloid maturation readily analyzed by FACS analysis, suggesting it could be an additional PD marker to be used in clinical studies. Indeed, it was found that SY-1425 induced CD38 cell surface expression at similar levels in RARA-high AML cell lines and the NB-4 APL cell line, but not in RARA-low cell lines. We also investigated combinations of SY-1425 with approved or investigational AML and MDS agents in in vitro and in vivo models to inform future clinical studies and to further explore potential PD markers unique to the combined action of the drugs. Several standard of care agents and drugs in current development were found to have synergistic interactions with SY-1425 in RARA-high but not RARA-low cell lines. In particular, azacitidine and decitabine each showed strong in vitro synergy with SY-1425. Evaluation of SY-1425 plus azacitidine in a RARA-high PDX model of non-APL AML demonstrated a better response compared to either agent alone. Additional genome-wide ChIP-seq and expression studies of RARA-high cells treated with various combinations are being investigated to identify optimal PD markers for these combinations. These studies support the use of DHRS3 mRNA induction in tumor cells as a PD marker in the recently initiated Ph2 study of SY-1425 in genomically-defined non-APL AML and MDS patients (clinicaltrials.gov NCT02807558) and further exploration as a PD marker for future combination studies. Disclosures McKeown: Syros Pharmaceuticals: Employment, Equity Ownership. Fiore:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

Retinoic acid receptor alpha (RARα) regulates myeloid differentiation and proliferation through the regulation of specific sets of genes. When unbound by a ligand, RARα is a repressive transcription factor while in its ligand-bound state it functions as a transcriptional activator. Previously, blast cells from a subset of individuals with non-APL AML were found to have a super-enhancer (SE), as revealed by H3K27 acetyl ChIP-Seq, associated with the RARA locus (hereafter called RARA-high), suggesting that tumor cell proliferation may have a dependency on RARA that can be exploited for therapeutic benefit. SEs are exceptionally large, highly active chromatin regions that are densely occupied by transcription factors and have been implicated in oncogene expression. Indeed, RARA-high non-APL AML cell lines showed >1000-fold increased sensitivity compared to RARA-low cells to the potent and selective RARα agonist SY-1425 (tamibarotene) as well as efficacy in non-APL AML patient derived xenograft models with a dependency on RARA. Since RARα is a transcription factor and the direct target of SY-1425, we investigated the change SY-1425 had on the transcriptional program of non-APL AML cell lines and the mechanism underlying those changes. Expression profiling on a panel of AML cell lines revealed that RARA-high AML cell lines had profound transcriptional changes in response to SY-1425, with 437 genes significantly changed, while RARA-low cell lines did not show significant gene expression changes. Gene set enrichment analysis (GSEA) of three RARA-high AML cell lines revealed that the genes upregulated by SY-1425 in the RARA-high cells are associated with immune signaling, interferon induction, protein secretion, and pathways associated with complement, MHC and integrin functions, all pathways indicative of more differentiated blood cells. Signatures downregulated by SY-1425 include MYC target genes. These findings are consistent with SY-1425 increasing the expression of genes involved in differentiation and decreasing those involved in proliferation. Genome-wide ChIP-Seq analysis revealed an increase in H3K27 acetylation at loci found to have strong RARα peaks as well as increased expression of those genes upon treatment with SY-1425. Together, these data support a model in which RARα binding nucleates functional enhancers in response to SY-1425 thereby upregulating proximal target gene expression and promotion of differentiation. The gene expression and epigenomic responses of RARA-high AML cell lines to SY-1425 were found to be similar to the responses of an APL cell line (NB-4) to retinoids or SY-1425. Gene sets identified in response to either retinoid treatment or genetic perturbation, such as forced expression or RAR-fusions or knockdown, matched the gene sets identified in RARA-high AML cell lines. Furthermore, the quantitative response of both NB-4 and RARA-high AML cell lines to SY-1425 was found to be similar. Across the genome, RARα binding was highly conserved between NB-4 and RARA-high AML cell lines with less overlap with the RARA-low cell lines. For example, the transcriptional and H3K27 acetylation alterations at the known PML-RARα target gene TGM2 following retinoid treatment was similar in NB-4 and the RARA- high cell lines. This locus also had a strong RARα binding site that is conserved among the cell lines and co-localized with a strong H3K27 acetylation peak. Consistent with the pattern of occupancy of RARα on the genome, the transcriptional response of the RARA enhancer-high cell lines to SY-1425 treatment was similar to the response of APL ex-vivo patient samples to retinoic acid treatment. These data support a model of a common biological response to retinoids between cells with the RARA-PML translocation in APL and cells with the RARA SE in AML. The mechanistic studies described here support the therapeutic potential of SY-1425 in myeloid leukemia patients who have a SE associated with RARA. A biomarker directed clinical trial of SY-1425, a potent and selective RARα agonist, in a subset of AML and MDS patients with an altered RARA locus (clinicaltrials.gov, NCT02807558) is supported by these data. Disclosures Fiore: Syros Pharmaceuticals: Employment, Equity Ownership. McKeown:Syros Pharmaceuticals: Employment, Equity Ownership. Lee:Syros Pharmaceuticals: Employment, Equity Ownership. Eaton:Syros Pharmaceuticals: Employment, Equity Ownership. Fritz:Syros Pharmaceuticals: Employment, Equity Ownership.

Precise DNA replication is crucial for genome maintenance, yet this process has been inherently difficult to study on a genome-wide level in untransformed differentiated metazoan cells. To determine how metazoan DNA replication can be repressed, we examined regions selectively under-replicated in Drosophila polytene salivary glands, and found they are transcriptionally silent and enriched for the repressive H3K27me3 mark. In the first genome-wide analysis of binding of the origin recognition complex (ORC) in a differentiated metazoan tissue, we find that ORC binding is dramatically reduced within these large domains, suggesting reduced initiation as one mechanism leading to under-replication. Inhibition of replication fork progression by the chromatin protein SUUR is an additional repression mechanism to reduce copy number. Although repressive histone marks are removed when SUUR is mutated and copy number restored, neither transcription nor ORC binding is reinstated. Tethering of the SUUR protein to a specific site is insufficient to block replication, however. These results establish that developmental control of DNA replication, at both the initiation and elongation stages, is a mechanism to change gene copy number during differentiation., National Institutes of Health (U.S.) (Grant GM57960), American Cancer Society. Research Professor Grant, National Institutes of Health (U.S.) (Grant 1U01HG004279)

Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide1, 2 has successfully identified specific subtypes of regulatory elements3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb response elements4, chromatin states5, transcription factor binding sites6, 7, 8, 9, RNA polymerase II regulation8 and insulator elements10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome on the basis of more than 300 chromatin immunoprecipitation data sets for eight chromatin features, five histone deacetylases and thirty-eight site-specific transcription factors at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and validated a subset of predictions for promoters, enhancers and insulators in vivo. We identified also nearly 2,000 genomic regions of dense transcription factor binding associated with chromatin activity and accessibility. We discovered hundreds of new transcription factor co-binding relationships and defined a transcription factor network with over 800 potential regulatory relationships.

The origin recognition complex (ORC) binds to the specific positions on chromosomes that serve as DNA replication origins. Although ORC is conserved from yeast to humans, the DNA sequence elements that specify ORC binding are not. In particular, metazoan ORC shows no obvious DNA sequence specificity, whereas yeast ORC binds to a specific DNA sequence within all yeast origins. Thus, whereas chromatin must play an important role in metazoan ORC's ability to recognize origins, it is unclear whether chromatin plays a role in yeast ORC's recognition of origins. This study focused on the role of the conserved N-terminal bromo-adjacent homology domain of yeast Orc1 (Orc1BAH). Recent studies indicate that BAH domains are chromatin-binding modules. We show that the Orc1BAH domain was necessary for ORC's stable association with yeast chromosomes, and was physiologically relevant to DNA replication in vivo. This replication role was separable from the Orc1BAH domain's previously defined role in transcriptional silencing. Genome-wide analyses of ORC binding in ORC1 and orc1bahΔ cells revealed that the Orc1BAH domain contributed to ORC's association with most yeast origins, including a class of origins highly dependent on the Orc1BAH domain for ORC association (orc1bahΔ-sensitive origins). Orc1bahΔ-sensitive origins required the Orc1BAH domain for normal activity on chromosomes and plasmids, and were associated with a distinct local nucleosome structure. These data provide molecular insights into how the Orc1BAH domain contributes to ORC's selection of replication origins, as well as new tools for examining conserved mechanisms governing ORC's selection of origins within eukaryotic chromosomes.

The origin recognition complex (ORC) specifies replication origin location. The Saccharomyces cerevisiae ORC recognizes the ARS (autonomously replicating sequence) consensus sequence (ACS), but only a subset of potential genomic sites are bound, suggesting other chromosomal features influence ORC binding. Using high-throughput sequencing to map ORC binding and nucleosome positioning, we show that yeast origins are characterized by an asymmetric pattern of positioned nucleosomes flanking the ACS. The origin sequences are sufficient to maintain a nucleosome-free origin; however, ORC is required for the precise positioning of nucleosomes flanking the origin. These findings identify local nucleosomes as an important determinant for origin selection and function.

AML is a highly genotypically and phenotypically heterogeneous cancer with many subtypes of the disease currently lacking clear targeted therapies. While several genetic alterations currently serve to stratify patients into prognostic subgroups and guide treatment decisions, the classification and treatment options for AML patients remain unsatisfactory. Numerous genome-wide analyses have provided evidence linking recurring genetic mutations in de novo AML to perturbations in epigenomic signaling, transcriptional regulation, and post-transcriptional mRNA processing, indicating that misregulation of gene expression is a key molecular feature of AML pathogenesis yet the diagnostic value and clinical utility of these findings remain unclear. Recently, a novel class of densely clustered cis-regulatory elements termed super-enhancers have emerged as key effectors initiating and maintaining cell type-specific gene expression in a variety of physiological and disease settings, including cancer. Tumor-specific super-enhancers regulate expression of key oncogenes and other cancer-essential genes, providing a novel strategy for detecting both known and unrecognized cancer dependencies of high diagnostic and therapeutic value. Here we describe the discovery and characterization of super-enhancer domains across a large cohort of AML patients and relevant normal hematopoietic stem and progenitor cell controls. To this end, we profiled over 60 primary samples at both the level of epigenomic activity and transcript abundance and identify transcriptionally hyperactive regions in AML patients compared to normal healthy donors. By combining patient-specific super-enhancer landscapes with gene expression profiles, we elucidate the transcription factor repertoires controlling chromatin dynamics and lineage specification in cellular derivatives of normal hematopoietic development and purified AML samples. Further analyses yield a striking delineation of disease-unique transcriptional subtypes which are specified by distinct patterns of super-enhancers at well-known AML-related genes such as HOXA9, MEIS1, ETV6, and HOXB3. In addition, we identify clusters of AML cases with shared super-enhancer domains suggesting convergence on common key drivers of AML. Together, our results provide the biological and disease relevance of super-enhancers in the context of normal and tumor cell states and demonstrate how the comprehensive mapping of gene regulatory elements elucidate clinically relevant subtypes and new therapeutic targets in AML. Citation Format: Matthew Eaton, Ryan Corces-Zimmerman, Jeremy Lopez, Christian Fritz, Eric Olson, Ravindra Majeti, Jakob Loven. Super-enhancer landscapes specify molecular subtypes and novel targets in acute myeloid leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2015-2212

Breast cancer cell state is driven by genomic mutational events, aberrant signaling cascades and transcriptional misregulation. The profound clinical impact of ER-modulatory drugs underscores the importance of understanding and targeting the transcriptional dependencies of breast cancer cells in an effort to create new therapeutics. Recently, large clusters of cis-acting enhancers, termed Super-enhancers, have emerged as dominant transcriptional regulators of oncogenes and other key tumor drivers in cancer cells, indicating that these elements control and maintain breast tumor cell state. Mapping these elements and the genes they control in patient tumors can provide insight into breast cancer classification and identify therapeutic targets. We have discovered Super-enhancers in breast cancer patient tissue and have characterized their roles in enforcing tumor cell state. We find that Super-enhancers define known clinical subgroups in invasive ductal carcinoma and pinpoint the putative transcriptional drivers of each subgroup. Super-enhancer-associated genes encode both known and novel therapeutic targets including kinases, phosphatases, chromatin regulators and transmembrane proteins, including key drivers such as ERRB2 in HER2+ patient samples, ESR1 in estrogen receptor positive samples, and CCND1 in samples of luminal subtype. shRNA knockdown confirms that Super-enhancer regulated genes are indeed essential for tumor cell survival and that Super-enhancer analysis in primary patient samples can be used to define new subtype specific cancer vulnerabilities. In sum, we describe the biological and disease relevance of Super-enhancer-associated genes in the context of tumor cell state, genome mutational landscape and drug target discovery. Citation Format: Cindy Collins, Mei Wei Chen, Matthew Eaton, David Orlando, Michael McKeown, Christian Fritz, Eric Olson, Matthew Guenther. Super-enhancers define breast cancer subclasses and identify novel tumor cell vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3837. doi:10.1158/1538-7445.AM2015-3837

Objective: To explore the role of chromatin conformation in the decline of cognition that accompanies aging and aging-related neurodegenerative diseases such as Alzheimer9s disease (AD). Background The plasticity of the epigenome suggests that it may be one medium through which life experiences throughout the life course could effect stable changes in gene expression that could ultimately influence the risk of dementia. Design/Methods: We base this investigation on a unique bank of frozen brains from subjects with longitudinal cognitive data collected in two studies of aging: the Memory and Aging Project and the Religious Order Study. A sample of dorsolateral prefrontal cortex was obtained from each of 759 subjects and using the Illumina Humanmet450K platform; this generated data on the extent of DNA methylation at 486,428 CpG sites distributed throughout the genome. Results: These data are remarkably similar across individuals, with over 98% pairwise correlation between any two subjects. However, using principal component (PC) analysis, we do find strong correlations with age (PC7) and sex (PC9) methylome-wide. Adjusting for these effects and other pertinent covariates, we find that the 319 subjects with AD at the time of death have higher levels of DNA methylation in many sites throughout the genome. However, the difference in the extent of methylation in the tissue sample at any given CpG site is modest. In the current model, the best result is returned by CpG1211454 which has a p=1.84x10^-9 in the AD vs. non-AD analysis, which exceeds our threshold of methylome-wide significance (p Conclusions: Our study provides evidence for loci where the extent of DNA methylation correlates with a diagnosis of AD, suggesting that the brain9s transcriptional potential may play a role in aging-related diseases such as dementia. Disclosure: Dr. De Jager has received personal compensation for activities with Merck Serono and Teva Neuroscience as a consultant.Dr. De Jager has received research support from Biogen Idec. Dr. Srivastava has nothing to disclose. Dr. Eaton has nothing to disclose. Dr. Chibnik has nothing to disclose. Dr. Kellis has nothing to disclose. Dr. Bennett has received personal compensation for activities with Danone, Inc., Dr. Wilmar Schwabe GmbH & Co, KG Pharmaceuticals, Eli Lilly & Company as a consultant. Dr. Bennett has received research support from Danone, Inc.

Using cDNA copies of transcripts and corresponding genomic sequences from the Berkeley Drosophila Genome Project, a set of 24,753 donor and acceptor splice sites were computed with a scanning algorithm that tested for single nucleotide insertion, deletion and substitution polymorphisms. Using this dataset, we developed a progressive partitioning approach to examining the effects of challenging the spliceosome system. Our analysis shows that information content increases near splice sites flanking progressively longer introns and exons, suggesting that longer splice elements require stronger binding of spliceosome components. Information also increases at splice sites near very short introns and exons, suggesting that short splice elements have crowding problems. We observe that the information found at individual splice sites depends upon a balance of splice element lengths in the vicinity, including both flanking and non-adjacent introns and exons. These results suggest an interdependence of multiple splicing events along the pre-mRNA, which may have implications for how the macromolecular spliceosome machine processes sets of neighboring splice sites.